G protein-coupled receptor (GPCR) drug development has traditionally focused on the concepts of orthosteric agonism and antagonism, in which receptor structure determines the nature of the downstream signal and ligand efficacy determines its intensity. Over the past decade, the newer paradigms of 'pluridimensional efficacy' and 'functional selectivity' have established that GPCRs mediate biological effects by engaging both classical G protein-dependent and novel G protein-independent signaling networks, and that ligand structure can 'bias' signaling by stabilizing active receptor states in different proportions than the native ligand. Such 'biased agonists' are novel pharmacological entities with the ability to qualitatively change GPCR signaling, in effect creating 'new receptors with distinct efficacy profiles driven by ligand structure. The promise of biased agonism resides with this ability to engender 'mixed' effects, and work during the current term of this project has demonstrated that arrestin-pathway selective agonism can produce potentially beneficial effects in vivo that are not attainable with conventional agonists or antagonists. At the same time, our data suggest that activating GPCRs in 'unnatural' ways can lead to unpredictable downstream biological consequences, especially when using ligands that selectively activate as-yet poorly characterized arrestin-dependent signaling networks. Efforts to develop biased therapeutics are thus hampered by an inability to predict the in vivo actions of arrestin-biased ligands based on their in vitro efficacy. The central hypothesis of this proposal is that the in vivo consequences arrestin-dependent signaling arises from activation of a relatively discrete and conserved set of biological responses that can be profiled in vitro using cell-based assays that are amenable to drug discovery research. During the next term, we plan to test this hypothesis using state-of-the-art biophysical and biochemical approaches combined with in vivo characterization of biased ligand action. Using intramolecular FlAsH BRET sensors that monitor conformational changes in arrestins following receptor activation, Aim 1 will examine the factors that determine the arrestin 'conformational signature' and determine the relationship between arrestin conformation and receptor/ligand efficacy in vitro. Using multi-dimensional efficacy profiling and SILAC-based whole cell phosphoproteomic analysis of signaling networks, Aim 2 will determine the range of biased ligand effects attainable in vitro and test whether the arrestin-dependent signaling network is conserved between different GPCRs. Aim 3 will then 'close the loop' by determining the relationship between arrestin conformation, in vitro ligand efficacy, and the biological phenotype associated with in vivo exposure to arrestin-selective biased agonists. These experiments will employ biased PTH1 parathyroid hormone receptor ligands in a murine model of bone formation. Completion of this project will help establish a rational framework for relating in vitro ligand efficacy to the in vivo actions of arrestin pathway-selective biased GPCR ligands, providing information and tools that are critical to the development of novel biased therapeutics.